Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [N-15,H-1] heteronuclear NMR spectroscopy

The interaction of yeast iso-1-cytochrome c with its physiological redox pa rtner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both N-15 and H-1 nuclei aris ing from the interaction of isotopically enriched N-15 cytochrome c with cy tochrome c peroxidase have been observed. For the diamagnetic, ferrous cyto chrome c, 34 amides are affected by binding, corresponding to residues at t he front face of the protein and in agreement with the interface observed i n the 1:1 crystal structure of the complex. In contrast, for the paramagnet ic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentall y observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated wi th H-N nuclei as inputs. In contrast to an earlier study, the results indic ate that there is no change in the geometry of the magnetic axes for cytoch rome c upon binding to cytochrome c peroxidase. This leads us to conclude t hat the additional effects observed for the ferric protein arise either fro m a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.